The molecular events underlying reciprocal CFTR-ENaC coupling, two major transport proteins implicated in pathophysiology of CF, is poorly understood. Revealing the mechanisms of this interaction will have potential ramifications in understanding the pathogenesis of CF. The main purpose of this application is to define the mechanisms by which CFTR interacts and modulates ENaC activity. Aim I will test the hypothesis that CFTR influences ENaC activity by means of a direct intermolecular interaction. We will determine the CFTR-ENaC interaction using Fluorescence Resonance Energy Transfer (FRET) microscopy and co-immunoprecipitation (co-IP). FRET is an approach that allows distance determination between CFTR and ENaC with near angstrom resolution, a distance sufficient for molecular interactions to occur. Our preliminary findings, using FRET, place the CFTR N-terminus and 1, 2, or 3 ENaC C-termini in sufficiently close proximity at the plasma membrane for a direct physical interaction to take place. FRET results are corroborated by both conventional co-IP and co-IP combined with fluorescence imaging. Our preliminary conventional co-IP has shown that untagged 2-ENaC interacts with untagged CFTR after transient transfection. Moreover, CFTR-ENaC association will be detected using the cell lines 16HBE14o- and LIM1864, which endogenously express both proteins. Also, Fluorescent spectroscopy will be used for a quantitative measure of the interaction. Aim II will define the specific binding site(s) within intracellular termini of 1 and 2 ENaC subunits responsible for their interaction with the termini of CFTR. As 1 ENaC is required to form a functional channel, and mice overexpressing 2 ENaC develop CF like lung disease, we will examine the interaction between these subunits and CFTR. In the first instance we will use a GST-fusion protein of the N-terminal tail of CFTR (GST-N-CFTR) to pull down ENaC subunits from cells transiently expressing individual epitope tagged ENaC subunits (1-Myc or 2-HA). If GST-N-CFTR fails to pull down any ENaC subunits, NBD1, R-domain, and the C-terminal tail of CFTR will be used. After identifying the CFTR cytoplasmic domains able to pull down intracellular tails of ENaC subunits, the site of interaction will be determined by successive deletions of the respective ENaC cytoplasmic tails. These experiments will be complemented by a pairwise binding assay. These results will help to map out the potential binding sites in CFTR and ENaC. Also, the outcome of this aim will shape the interaction model for CFTR-ENaC interaction. The findings from this proposal will provide a mechanistic view of how CFTR and ENaC interact with each other, and how their communication contributes to the pathogenesis of CF. Information resulting from this proposal might help to establish a new ways to correct electrolyte abnormalities in CF and pave the way to new rational approaches in CF therapy. PUBLIC HEALTH RELEVANCE Cystic Fibrosis (CF) is one of the most lethal genetic disorders that results from mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. CF affects approximately 30,000 children and adults in the US. Each year almost 2,500 babies are born with CF in the US alone. 1 in every 20 Americans is unaffected carrier of an abnormal CF gene. The mutations cause inadequate functioning of CFTR, which in turn leads to severe disruption in transport function of several epithelia in various organs. Affected organs include sweat glands, intestine, and the reproductive system, with the most devastating consequences when the disease affects airways. Gradual lung failure is the major life limiting factor in patients with CF. Decreased chloride secretion and increased salt absorption are the main contributing factors in the pathogenesis of CF. Decreased chloride secretion is a consequence of defective CFTR, and increased salt absorption results from the failure of CFTR to restrict salt absorption through a sodium channel (named the Epithelial Na+ Channel, ENaC). The mechanism by which CFTR controls adequate functioning of ENaC is still obscure, and uncovering the nature of this interaction between CFTR and ENaC is critical to our understanding of fluid and salt balance in patients suffering from CF. The findings of the present application will contribute to the basic knowledge of salt and fluid balance in the airways both under physiological and pathophysiological conditions, leading ultimately to a new avenue of therapeutic interventions.